Galleria mellonella (Greater Wax Moth) larvae are becoming an established disease model in biological research. The benefits of using Galleria are wide, ranging from the lack of ethical issues of using mammals, to the size and ease of handling, to the fact that they have a complex innate immune system like mammals (1). Galleria have cellular responses such as nodulisation and encapsulation, and haemolymph cells can phagocytose microbes (2, 3). It has also been found that the epithelial cells in the gut of the larvae share similar physiological phenotypes as the intestinal cells in the mammalian digestive system (3). Finally, the larvae can be incubated in temperatures up to 37° C., making it a suitable model for investigating diseases of humans.
G. mellonella are mainly farmed as food-stuffs for reptiles and amphibians (4) and purchased through suppliers or from local pet shops. As a consequence of the method of production, G. mellonella are not grown under standardised conditions and are exposed to conditions which influence the natural bacterial flora found on the larvae, which subsequently may have an effect on the susceptibility of larvae to disease (3).
The inventors have found batch-to-batch variability with G. mellonella larvae purchased from different suppliers. With some batches, significant numbers of larvae died in control groups dosed only with PBS (phosphate buffered saline), because the act of injecting PBS resulted in transfer of the microbial flora into the body cavity. Control group failures occur in around 30% of bait shop G. mellonella larvae. Similar problems are experienced with other research organisms such as Manduca sexta and Caenorhabditis elegans. This limits the potential for widespread use of these organisms as a reliable research model.
Previous workers have sterilised the alimentary tract of Galleria mellonella larvae by immersing the larvae in an ethyl alcohol solution for 5-6 hours (AU3222878). Although the concentration of alcohol used by these workers was not disclosed, it must have been a low concentration in order for the larvae to survive the long incubation period. The method prepares the larvae so that extracts from them can be utilised in an animal immunisation process. The method was not used to reduce the microbial flora on the cuticle of the larvae.
CN103098762 disclosed sterilising mealworms with a high concentration (75%) ethanol solution for 5-15 seconds. A mealworm treated in such a way was then packaged and kept in order to obtain a pupa and, ultimately, an adult insect. There was no suggestion that the mealworms might be useful as research-grade organisms, for use in methods requiring a reliable control group in experiments involving injection through the cuticle.
Shadmehr et al. (2007; Pakistan J. Biol. Sci. vol. 10 p 2910-2914) discussed the use of a 70% ethanol solution to sterilise cyst nematodes, for subsequent infection of plant cells. The nematodes were treated with the ethanol for 1 minute and subsequently treated with sodium hypochlorite and Triton X100. The conditions disclosed in the publication would be lethal to Galleria mellonella larvae.
Togashi (2004; J. Econ. Entomol. vol. 97 p 941-945) disclosed “dipping” beetle larvae into 70% and 99% ethanol. There was no indication of the time period used and there was no suggestion that the larvae might be useful as research-grade organisms, for use in methods requiring a reliable control group in experiments involving injection through the cuticle. The larvae were used as a host for nematode infection.
There remains a need to provide research-grade invertebrate organisms, especially insect larvae.